DESCRIPTION (taken from the application): Caldesmon (CaD) plays a dual role during smooth muscle contraction: Structurally, it helps to organize the thick and thin filaments by binding to both actin and myosin, and functionally, it modulates crossbridge cycling by reversibly inhibiting the actomyosin interaction. How CaD is regulated remains unclear. Our long-term goal is to understand the thin filament-based regulation of smooth muscle contraction and how it interacts with the well-established thick filament-based regulation. In this proposal, efforts will be focused on the elucidation of the regulatory mechanism of smooth muscle Cal), and to test the hypothesis that CaD is needed for smooth muscle to function normally. Specifically, we will (1) test whether CaD is able to tether myosin filaments to actin without impeding the sliding movement by characterizing the nature of CaD binding to actin and myosin using analytical ultracentrifugation and isothermal titration calorimetry, and by determining the residues involved in binding with chemical crosslinking; (2) test whether CaD exerts its action via conformational changes by obtaining structural information of CaD using fluorescence resonance energy transfer and NMR spectroscopy with segmental isotope labeling; (3) test whether the function of CaD is regulated by phosphorylation by assessing the significance of in vivo phosphorylation of CaD using mass spectrometry and anti-phosphopeptide antibodies; and (4) test whether removal of CaD affects the animal's viability or its smooth muscle contractility by gene knockout experiments and, if abnormalities observed, rescue by expressing wild-type or mutated Cal). Information generated from these studies will afford a better understanding of the thin filament-based regulatory mechanism, which may be crucial for proper diagnosis and future development of therapeutic measures of many smooth muscle related diseases.